Shao-Chen Sun

Small GTPase RhoA regulates cytoskeleton dynamics during porcine oocyte maturation and early embryo development

Mammalian oocyte maturation is distinguished by asymmetric division that is regulated primarily by cytoskeleton, including microtubules and microfilaments. Small Rho GTPase RhoA is a key regulator of cytoskeletal organization which regulates cell polarity, migration, and division. In this study, we investigated the roles of RhoA in mammalian oocyte meiosis and early embryo cleavage. (1) Disrupting RhoA activity or knock down the expression of RhoA caused the failure of polar body emission. This may have been due to decreased actin assembly and subsequent spindle migration defects. The involvement of RhoA in this process may have been though its regulation of actin nucleators ROCK, p-Cofilin, and ARP2 expression. (2) In addition, spindle morphology was also disrupted and p-MAPK expression decreased in RhoA inhibited or RhoA KD oocytes, which indicated that RhoA also regulated MAPK phosphorylation for spindle formation. (3) Porcine embryo development was also suppressed by inhibiting RhoA activity. Two nuclei were observed in one blastomere, and actin expression was reduced, which indicated that RhoA regulated actin-based cytokinesis of porcine embryo. Thus, our results demonstrated indispensable roles for RhoA in regulating porcine oocyte meiosis and cleavage during early embryo development.

Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation

Deoxynivalenol (DON) is a widespread trichothecene mycotoxin which contaminates agricultural staples and elicits a complex spectrum of toxic effects on humans and animals. It has been shown that DON impairs oocyte maturation, reproductive function and causes abnormal fetal development in mammals; however, the mechanisms remain unclear. In the present study, we investigate the possible reasons of the toxic effects of DON on porcine oocytes. Our results showed that DON significantly inhibited porcine oocyte maturation and disrupted meiotic spindle by reducing p-MAPK protein level, which caused retardation of cell cycle progression. In addition, up-regulated LC3 protein expression and aberrant Lamp2, LC3 and mTOR mRNA levels were observed with DON exposure, together with Annexin V-FITC staining assay analysis, these results indicated that DON treatment induced autophagy/apoptosis in porcine oocytes. We also showed that DON exposure increased DNA methylation level in porcine oocytes through altering DNMT3A mRNA levels. Histone methylation levels were also changed showing with increased H3K27me3 and H3K4me2 protein levels, and mRNA levels of their relative methyltransferase genes, indicating that epigenetic modifications were affected. Taken together, our results suggested that DON exposure reduced porcine oocytes maturation capability through affecting cytoskeletal dynamics, cell cycle, autophagy/apoptosis and epigenetic modifications.

Rho-GTPase Effector ROCK Phosphorylates Cofilin in Actin-Meditated Cytokinesis During Mouse Oocyte Meiosis

ABSTRACT During oocyte meiosis, a spindle forms in the central cytoplasm and migrates to the cortex. Subsequently, the oocyte extrudes a small body and forms a highly polarized egg; this process is regulated primarily by actin. ROCK is a Rho-GTPase effector that is involved in various cellular functions, such as stress fiber formation, cell migration, tumor cell invasion, and cell motility. In this study, we investigated possible roles for ROCK in mouse oocyte meiosis. ROCK was localized around spindles after germinal vesicle breakdown and was colocalized with cytoplasmic actin and mitochondria. Disrupting ROCK activity by RNAi or an inhibitor resulted in cell cycle progression and polar body extrusion failure. Time-lapse microscopy showed that this may have been due to spindle migration and cytokinesis defects, as chromosomes segregated but failed to extrude a polar body and then realigned. Actin expression at oocyte membranes and in cytoplasm was significantly decreased after these treatments. Actin caps were also disrupted, which was confirmed by a failure to form cortical granule-free domains. The mitochondrial distribution was also disrupted, which indicated that mitochondria were involved in the ROCK-mediated actin assembly. In addition, the phosphorylation levels of Cofilin, a downstream molecule of ROCK, decreased after disrupting ROCK activity. Thus, our results indicated that a ROCK-Cofilin-actin pathway regulated meiotic spindle migration and cytokinesis during mouse oocyte maturation.

Effect of mycotoxin-containing diets on epigenetic modifications of mouse oocytes by fluorescence microscopy analysis.

Mycotoxins, such as aflatoxin (AF), fumonisin B1, zearalenone (ZEA), and deoxynivalenol (DON), are commonly found in many food commodities. Mycotoxins have been shown to increase DNA methylation levels in a human intestinal cell line. We previously showed that the developmental competence of oocytes was affected in mice that had been fed a mycotoxin-containing diet. In this study, we explored possible mechanisms of low mouse oocyte developmental competence after mycotoxin treatment in an epigenetic modification perspective. Mycotoxin-contaminated maize (DON at 3,875 μg/kg, ZEA at 1,897 μg/kg, and AF at 806 μg/kg) was included in diets at three different doses (mass percentage: 0, 15, and 30%) and fed to mice for 4 weeks. The fluorescence intensity analysis showed that the general DNA methylation levels increased in oocytes from high dose mycotoxin-fed mice. Mouse oocyte histone methylation was also altered. H3K9me3 and H4K20me3 level increased in oocytes from mycotoxin-fed mice, whereas H3K27me3 and H4K20me2 level decreased in oocytes from mycotoxin-fed mice. Thus, our results indicate that naturally occurring mycotoxins have effects on epigenetic modifications in mouse oocytes, which may be one of the reasons for reduced oocyte developmental competence.

Sirt3 prevents maternal obesity-associated oxidative stress and meiotic defects in mouse oocytes

Maternal obese environment has been reported to induce oxidative stress and meiotic defects in oocytes, however the underlying molecular mechanism remains unclear. Here, using mice fed a high fat diet (HFD) as an obesity model, we first detected enhanced reactive oxygen species (ROS) content and reduced Sirt3 expression in HFD oocytes. We further observed that specific depletion of Sirt3 in control oocytes elevates ROS levels while Sirt3 overexpression attenuates ROS production in HFD oocytes, with significant suppression of spindle disorganization and chromosome misalignment phenotypes that have been reported in the obesity model. Candidate screening revealed that the acetylation status of lysine 68 on superoxide dismutase (SOD2K68) is dependent on Sirt3 deacetylase activity in oocytes, and acetylation-mimetic mutant SOD2K68Q results in almost threefold increase in intracellular ROS. Moreover, we found that acetylation levels of SOD2K68 are increased by ∼80% in HFD oocytes and importantly, that the non-acetylatable-mimetic mutant SOD2K68R is capable of partially rescuing their deficient phenotypes. Together, our data identify Sirt3 as an important player in modulating ROS homeostasis during oocyte development, and indicate that Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress and meiotic defects in oocytes under maternal obese conditions.

Aflatoxin B1 is toxic to porcine oocyte maturation

As a toxic secondary metabolite of Aspergillus species, Aflatoxin B1 (AFB1) is a major food and feed contaminant in tropical and sub-tropical regions with high temperature and humidity. It has been reported to be toxic to the female reproductive system in laboratory and domestic animals. In the present study, the influence of acute exposure to AFB1 (10 and 50 μM, 44h) on porcine oocyte maturation and its possible mechanism were investigated. The maturation rates of oocytes decreased significantly in the presence of 50 μM of AFB1. Cell cycle analysis showed that most oocytes were arrested at germinal vesicle breakdown or meosis I stage. However, actin assembly, spindle structure and chromosome alignment were not disrupted after exposure to 50 μM AFB1. Further study showed that DNA methylation levels increased in treated oocytes (50 μM). Histone methylation levels were also analysed after treatment (50 μM): H3K27me3 and H3K4me2 levels decreased, whereas H3K9me3 level increased, indicating that epigenetic modification was affected. AFB1 treatment (50 μM) also induced oxidative stress and further led to autophagy, as shown by accumulation of reactive oxygen species, up-regulated LC3 protein expression and increased mRNA levels of ATG3, ATG5 and ATG7. Annexin V-FITC staining assay revealed that AFB1 treatment (50 μM) resulted in oocyte early apoptosis, which was confirmed by increased Bak, Bax, Bcl-xl mRNA levels. Collectively, our results suggest that AFB1 disrupts porcine oocyte maturation through changing epigenetic modifications as well as inducing oxidative stress, excessive autophagy and apoptosis.

ROCK inhibitor Y-27632 prevents porcine oocyte maturation

The inhibitor Y-27632 is a specific selective inhibitor of Rho-associated protein kinases (ROCKs), which are downstream effectors of Rho guanosine triphosphatease (GTPases) and regulate Rho-associated cellular functions, including actin cytoskeletal organization. Little is known regarding the effects of Y-27632 on mammalian oocyte maturation. In the present study, we investigated the effects of Y-27632 on porcine oocyte meiosis and possible regulatory mechanisms of ROCK during porcine oocyte maturation. We found that ROCK accumulated not only at spindles, but also at the cortex in porcine oocytes. Y-27632 treatment reduced ROCK expression, and inhibited porcine oocyte meiotic maturation, which might be because of the impairment of actin expression and actin-related spindle positioning. Y-27632 treatment also disrupted the formation of actin cap and cortical granule-free domain, which further confirmed a spindle positioning failure. Thus, Y-27632 has significant effects on the meiotic competence of mammalian oocytes by reducing ROCK expression, and the regulation is related to its effects on actin-mediated spindle positioning.

Rab5a is required for spindle length control and kinetochore-microtubule attachment during meiosis in oocytes

Rab GTPases are highly conserved components of vesicle trafficking pathways. Rab5, as a master regulator of endocytic trafficking, has been shown to function in membrane tethering and docking. However, the function of Rab5 in meiosis has not been addressed. Here, we report elongated spindles and misaligned chromosomes, with kinetochore‐microtubule misattachments, on specific depletion of Rab5a in mouse oocytes. Moreover, the localization and levels of centromere protein F (CENPF), a component of the nuclear matrix, are severely reduced at kinetochores in metaphase oocytes following Rab5a knockdown. Consistent with this finding, nuclear lamina disassembly in the transition from prophase arrest to meiosis I is also impaired in Rab5a‐depleted oocytes. Notably, oocyte‐specific ablation of CENPF phenocopies the meiotic defects resulting from Rab5a knockdown. In summary, our data support a model where Rab5a‐positive vesicles, likely through interaction with nuclear lamina, modulate CENPF localization and levels at centromeres, consequently ensuring proper spindle length and kinetochore‐microtubule attachment in meiotic oocytes.—Ma, R., Hou, X., Zhang, L., Sun, S.‐C., Schedl, T., Moley, K., Wang, Q. Rab5a is required for spindle length control and kinetochore‐microtubule attachment during meiosis in oocytes. FASEB J. 28, 4026‐4035 (2014). www.fasebj.org

Acrylamide toxic effects on mouse oocyte quality and fertility in vivo

Acrylamide is an industrial chemical that has attracted considerable attention due to its presumed carcinogenic, neurotoxic, and cytotoxic effects. In this study we investigated possible acrylamide reproductive toxic effects in female mice. Mice were fed an acrylamide-containing diet for 6 weeks. Our results showed the following effects of an acrylamide-containing diet. (1) Ovary weights were reduced in acrylamide-treated mice and oocyte developmental competence was also reduced, as shown by reduced GVBD and polar body extrusion rates. (2) Acrylamide feeding resulted in aberrant oocyte cytoskeletons, as shown by an increased abnormal spindle rate and confirmed by disrupted γ-tubulin and p-MAPK localization. (3) Acrylamide feeding resulted in oxidative stress and oocyte early stage apoptosis, as shown by increased ROS levels and p-MAPK expression. (4) Fluorescence intensity analysis showed that DNA methylation levels were reduced in acrylamide-treated oocytes and histone methylation levels were also altered, as H3K9me2, H3K9me3, H3K4me2, and H3K27me3 levels were reduced after acrylamide treatment. (5) After acrylamide feeding, the litter sizes of acrylamide-treated mice were significantly smaller compared to thus of control mice. Thus, our results indicated that acrylamide might affect oocyte quality through its effects on cytoskeletal integrity, ROS generation, apoptosis induction, and epigenetic modifications.

Formin mDia1, a downstream molecule of FMNL1, regulates Profilin1 for actin assembly and spindle organization during mouse oocyte meiosis☆

Mammalian diaphanous1 (mDia1) is a homologue of Drosophila diaphanous and belongs to the Formin-homology family of proteins that catalyze actin nucleation and polymerization. Although Formin family proteins, such as Drosophila diaphanous, have been shown to be essential for cytokinesis, whether and how mDia1 functions during meiosis remain uncertain. In this study, we explored possible roles and the signaling pathway involved for mDia1 using a mouse oocyte model. mDia1 depletion reduced polar body extrusion, which may have been due to reduced cortical actin assembly. mDia1 and Profilin1 had similar localization patterns in mouse oocytes and mDia1 knockdown resulted in reduced Profilin1 expression. Depleting FMNL1, another Formin family member, resulted in reduced mDia1 expression, while RhoA inhibition did not alter mDia1 expression, which indicated that there was a FMNL1-mDia1-Profilin1 signaling pathway in mouse oocytes. Additionally, mDia1 knockdown resulted in disrupting oocyte spindle morphology, which was confirmed by aberrant p-MAPK localization. Thus, these results demonstrated indispensable roles for mDia1 in regulating mouse oocyte meiotic maturation through its effects on actin assembly and spindle organization.